Role of GSK-3beta activation and alpha7 nAChRs in Abeta(1-42)-induced tau phosphorylation in PC12 cells

β-amyloid peptide 1–42 (Aβ_1–42) and hyperphosphorylated tau are associated with neurodegeneration in Alzheimer's disease. Emerging evidence indicates that Aβ_1–42 can potentiate hyperphosphorylation of tau in cell lines and in transgenic mice, but the underlying mechanism(s) remains unclear. In this study, Aβ_1–42-induced tau phosphorylation was investigated in differentiated PC12 cells. Treatment of cells with Aβ_1–42 increased phosphorylation of tau at serine-202 as detected by AT8 antibody. This Aβ_1–42-induced tau phosphorylation paralleled phosphorylation of glycogen synthase kinase-3β (GSK-3β) at tyrosine-216 (GSK-3β-pY216), which was partially inhibited by the GSK-3β inhibitor, CHIR98023. Aβ_1–42-induced tau phosphorylation and increase in GSK-3β-pY216 phosphorylation were also partially attenuated by α7 nicotinic acetylcholine receptor (α7 nAChR) selective ligands including agonist A-582941 and antagonists methyllycaconitine and α-bungarotoxin. The α7 nAChR agonist and the GSK-3β inhibitor had no additive effect. These observations suggest that α7 nAChR modulation can influence Aβ_1–42-induced tau phosphorylation, possibly involving GSK-3β. This study provides evidence of nAChR mechanisms underlying Aβ_1–42 toxicity and tau phosphorylation, which, if translated in vivo , could provide additional basis for the utility of α7 nAChR ligands in the treatment of Alzheimer's disease.     

Monoclonal Antibodies Against the Human Metalloprotease EC 3.4.24.15 Label Neurofibrillary Tangles in Alzheimer's Disease Brain

Abstract: Alzheimer's disease is characterized neuropathologically by the presence of neuritic and amyloid plaques, vascular amyloid, and neurofibrillary tangles in specific brain areas. The main constituent of amyloid deposits is amyloid β protein, a 40–42 amino acid proteolytic product of the amyloid β-precursor protein. In our search for proteases that can generate the N-terminus of amyloid β protein (β-secretases), we discovered a thiol-dependent metalloprotease that was identified, by peptide sequencing, as metalloendopeptidase EC 3.4.24.15. In vitro, the metalloprotease cleaves the methionine-aspartic acid bond in a 10 amino acid synthetic peptide, indicating that it could generate the N-terminus of amyloid β protein, and generates amyloidogenic fragments from full-length recombinant amyloid β-precursor protein. Mouse monoclonal antibodies produced against a unique synthetic peptide from the metalloprotease labeled various monkey tissues as detected by western blots and immunohistochemistry. Unexpectedly, two monoclonal antibodies, IVD6 and IIIF3, immunolabeled strongly intracellular neurofibrillary tangles, neurites of senile plaques, and neuropil threads, but not "ghost" tangles or amyloid in sections taken from Alzheimer's disease brain. This finding provides further evidence for the metalloprotease's relevance to Alzheimer's disease pathology, although the connection between tangle staining and the formation of amyloid β protein remains to be elucidated.     

Fate of Cerebrospinal Fluid-Borne Amyloid β-Peptide: Rapid Clearance into Blood and Appreciable Accumulation by Cerebral Arteries

Abstract: In Alzheimer's disease, the neuritic or senile amyloid plaques in hippocampus and association cortex, the diffuse plaques in brain areas such as the cerebellum and sensorimotor cortex, and the amyloid deposits in the walls of pial and parenchymal blood vessels are mainly composed of amyloid β-peptides. In the present study, either soluble 40-residue amyloid β-peptide radiolabeled with ¹²⁵I (I-sAβ) or [¹⁴C]polyethylene glycol ([¹⁴C]-PEG, a reference material) was briefly infused into one lateral ventricle of normal rats. By 3.5 min, 30% of the I-sAβ was cleared from ventricular CSF into blood; another 30% was removed over the next 6.5 min. No [¹⁴C]PEG was lost from the CSF-brain system during the first 5 min, and only 20% was cleared by 10 min. Much of the I-sAβ that reached the subarachnoid space was retained by pial arteries and arterioles. Virtually no I-sAβ was found in brain. The clearance of amyloid β-peptides from the CSF-brain system, reported herein for normal rats, may be reduced in Alzheimer's disease, thus contributing to amyloid deposition in cerebral tissue and blood vessels.     

Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-β (1–42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators

Aggregation of amyloid-β (Aβ) peptides is a central phenomenon in Alzheimer's disease. Zn(II) and Cu(II) have profound effects on Aβ aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Aβ₄₂ fibrillization and initiate formation of non-fibrillar Aβ₄₂ aggregates, and that the inhibitory effect of Zn(II) (IC₅₀ = 1.8 μmol/L) is three times stronger than that of Cu(II). Medium and high-affinity metal chelators including metallothioneins prevented metal-induced Aβ₄₂ aggregation. Moreover, their addition to preformed aggregates initiated fast Aβ₄₂ fibrillization. Upon prolonged incubation the metal-induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Aβ₄₂. H13A and H14A mutations in Aβ₄₂ reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross-β core structure within region 10–23 of the amyloid fibril. Cu(II)-Aβ₄₂ aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)-Aβ₄₂ aggregates were non-toxic. Disturbed metal homeostasis in the vicinity of zinc-enriched neurons might pre-dispose formation of metal-induced Aβ aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal-chelating therapies for Alzheimer's disease is discussed in this light.     

Flavonoid-mediated presenilin-1 phosphorylation reduces Alzheimer's disease beta-amyloid production

Glycogen synthase kinase 3 (GSK-3) dysregulation is implicated in the two Alzheimer's disease (AD) pathological hallmarks: β-amyloid plaques and neurofibrillary tangles. GSK-3 inhibitors may abrogate AD pathology by inhibiting amyloidogenic γ-secretase cleavage of amyloid precursor protein (APP). Here, we report that the citrus bioflavonoid luteolin reduces amyloid-β (Aβ) peptide generation in both human 'Swedish' mutant APP transgene-bearing neuron-like cells and primary neurons. We also find that luteolin induces changes consistent with GSK-3 inhibition that ( i ) decrease amyloidogenic γ-secretase APP processing, and ( ii ) promote presenilin-1 (PS1) carboxyl-terminal fragment (CTF) phosphorylation. Importantly, we find GSK-3α activity is essential for both PS1 CTF phosphorylation and PS1-APP interaction. As validation of these findings in vivo , we find that luteolin, when applied to the Tg2576 mouse model of AD, decreases soluble Aβ levels, reduces GSK-3 activity, and disrupts PS1-APP association. In addition, we find that Tg2576 mice treated with diosmin, a glycoside of a flavonoid structurally similar to luteolin, display significantly reduced Aβ pathology. We suggest that GSK-3 inhibition is a viable therapeutic approach for AD by impacting PS1 phosphorylation-dependent regulation of amyloidogenesis.     

Protein Kinase FA/Glycogen Synthase Kinase 3α Predominantly Phosphorylates the In Vivo Sites of Ser502, Ser506, Ser603, and Ser666 in Neurofilament

Abstract: In this report, the phosphorylation sites of neurofilament protein of medium molecular mass (NF-M) by protein kinase F_A/glycogen synthase kinase 3α (kinase F_A/GSK-3α) were determined by two-dimensional electrophoresis/TLC, phosphoamino acid analysis, HPLC, Edman degradation, and peptide sequencing. Kinase F_A/GSK-3α phosphorylates NF-M predominantly on serine, residue. Three major tryptic phosphopeptide peaks were resolved by C₁₈ reverse-phase HPLC. Edman degradation and peptide sequence analysis revealed that AKS(p)PVSK is the phosphorylation site sequence for the first major peak. When mapping with the amino acid sequence of neurofilament, we finally demonstrate Ser⁶⁰³-Pro, one of the in vivo sites in NF-M, as the major site phosphorylated by kinase F_A/GSK-3α. By using the same approach, we also identified the in vivo sites of Ser⁵⁰²-Pro, Ser⁵⁰⁶-Pro, and Ser⁶⁶⁶-Pro as the other three major sites in NF-M phosphorylated by kinase F_A/GSK-3α. Taken together, the results provide initial evidence that kinase F_A/GSK-3α may represent a physiologically relevant protein kinase involved in the in vivo phosphorylation of NF-M. Because Ser⁵⁰², Ser⁵⁰⁶, Ser⁶⁰³, and Ser⁶⁶⁶ are all flanked by a carboxyl-terminal proline residue, the results provide further evidence that F_A/GSK-3α may represent a proline-directed protein kinase involved in the structure-function regulation of the neuronal cytoskeletal system.     

Protein Kinase FA/GSK-3 Phosphorylates on Ser235-Pro and Ser404-Pro that Are Abnormally Phosphorylated in Alzheimer's Disease Brain

Abstract— Previously, we identified protein kinase F_A/gly-cogen synthase kinase-3 (GSK-3) as a microtubule-associated protein kinase that can incorporate 4 mol of phosphates into 1 mol of protein and cause its electrophoretic mobility shift in sodium dodecyl sulfate gels, a unique property characteristic of paired helical filament-associated pathological (PHF-) in Alzheimer's disease brains. In this report, we identified TPPKS(p)PSAAK and SPVVSGDTS(p)PR as two phosphorylation site sequences phosphorylated by kinase F_A/GSK-3 in using peptide sequence analysis and sequential manual Edman degradation for radiosequencing. When mapping with human brain sequence, we further identified Ser²³⁵-Pro and Ser⁴⁰⁴-Pro as the two major phosphorylation sites according to the numbering of the longest isoform. Ser²³⁵ and Ser⁴⁰⁴ have been reported as two of the major abnormal phosphorylation sites in PHF-. Taken together, the results provide initial evidence that protein kinase F_A/GSK-3 may represent one of the Ser-Pro motif-directed kinases involved in the abnormal phosphorylation of pathological PHF- in Alzheimer's disease brain.     

APOE polymorphism and clinical duration determine regional neuropathology in Swedish APP(670, 671) mutation carriers: implications for late-onset Alzheimer's disease

Neurofibrillary changes throughout the brain were investigated for three relatives who carried the Swedish APP_670,671 mutation which causes overproduction of Aβ40 and Aβ42. They differed in terms of APOE genotype, age at the onset of dementia, and disease duration (P1: ɛ2/3, age 57, 11 years; P2: ɛ2/3, age 61, 5 years; P3: ɛ4/4, age 44, 12 years). For each subject, paraffin-embedded sections from diverse anatomically and cytoarchitectonically well-preserved regions were stained using the modified Bielschowsky method. Neurofibrillary tangles (NFT) and neuritic plaques (NP) were counted, and the area occupied by plaque estimated (%NP). In addition, sections from the medial frontal gyrus were stained with monoclonal antibodies to APOE. The regional patterns of neurofibrillary changes were consistent with those for late-onset AD. Longer disease duration was associated with further accumulations in earlier-affected areas, with superficial cortical layers consistently containing higher %NP than deep layers. APOE ɛ4/4 was associated with deeper limbic and frontal NFT, with an excess of NP (especially in the outer parietal cortex) which stained heavily for APOE - as well as with very early onset. APP_670,671 mutation carriers demonstrate regional brain neurofibrillary changes characteristic of late-onset Alzheimer's disease with evidence for more Aβ deposition for ɛ4/4 than ɛ2/3. This raises the possibility that early Braak Stage I-II lesions might also follow this pattern of promotion.     

Amyloid Angiopathy of Alzheimer''s Disease: Amino Acid Composition and Partial Sequence of a 4,200-Dalton Peptide Isolated from Cortical Microvessels

The cardinal lesions of Alzheimer's disease are neurofibrillary tangles, senile neuritic plaques, and vascular amyloid, the latter generally involving cortical arteries and small arterioles. All three lesions are composed of amyloid-like, beta-pleated sheet fibrils. Recently, a 4,200-dalton peptide has been isolated from extraparenchymal meningeal vessels, neuritic plaques, and neurofibrillary tangles. The assumption of N-terminal homogeneity in vascular amyloid has been used as an argument for a neuronal (versus blood) origin of the peptide. However, intracortical microvessels from Alzheimer's disease have not been previously isolated. The present studies describe the isolation of a microvessel fraction from Alzheimer's disease and control fresh autopsy human brain. Alzheimer's disease isolated brain microvessels that were extensively laden with amyloid and control microvessels were solubilized in 90% formic acid and analyzed by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The arteriole fraction from the Alzheimer's subject with extensive amyloid angiopathy contained a unique 4,200-dalton peptide, whereas the arterioles or capillaries isolated from two controls and two Alzheimer's disease subjects without angiopathy did not. This peptide was purified by HPLC and amino acid composition analysis showed the peptide is nearly identical to the 4,200-dalton peptide recently isolated from neuritic plaques or from neurofibrillary tangles. Sequence analysis revealed N-terminal heterogeneity. The N-terminal sequence was: Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr, which is identical to the N-terminal sequence of the 4,200-dalton peptide isolated previously from extraparenchymal meningeal vessels and neuritic plaques.(ABSTRACT TRUNCATED AT 250 WORDS)     

α2-Macroglobulin Attenuates β-Amyloid Peptide 1–40 Fibril Formation and Associated Neurotoxicity of Cultured Fetal Rat Cortical Neurons

Abstract: β-Amyloid peptides (Aβ) are deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimer's disease. The neurotoxicity of Aβ in cultured neurons is dependent on its aggregation state, but the factors contributing to aggregation and fibril formation are poorly understood. In the present study, we investigated whether α₂-macroglobulin (α₂M), a protein present in neuritic plaques and elevated in Alzheimer's disease brain, is a potential regulatory factor for Aβ fibril formation. Previous studies in our laboratory have shown that α₂M is an Aβ binding protein. We now report that, in contrast to another plaque-associated protein, α₁-antichymotrypsin, α₂M coincubated with Aβ significantly reduces aggregation and fibril formation in vitro. Additionally, cultured fetal rat cortical neurons are less vulnerable to the toxic actions of aged Aβ following pretreatment with α₂M. We postulate that α₂M is able to maintain Aβ in a soluble state, preventing fibril formation and associated neurotoxicity.     

Amyloid β Protein (25–35) Stimulation of Phospholipase C in LA-N-2 Cells

Abstract: The amyloid β protein (25–35) stimulated appearance of ³H-inositol phosphates from [³H]inositol-prelabeled LA-N-2 cells was investigated. This stimulation was unaltered by extra- and intracellular calcium chelators in a calcium-free medium or by several protein kinase inhibitors. This phospholipase C stimulation by amyloid β protein appeared to be pertussis toxin sensitive. It is possible that this phospholipase C stimulation by amyloid β protein is a receptor-mediated process. This possibility is based on two related observations. The stimulation is ablated by the presence of conventional antagonists for metabotropic, adrenergic, and bombesin agonists. The IC₅₀ values were 12 µ M for propranolol, 15 µ M for AP-3, and 25 n M for [Tyr⁴, d -Phe¹²]bombesin. Additional support comes from results of densensitization and resensitization experiments. Amyloid β protein stimulation of phospholipase C was absent from LA-N-2 cells previously treated with norepinephrine, trans -1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), bombesin, or amyloid β peptide. In a similar manner, LA-N-2 cells previously treated with amyloid β protein were no longer responsive to norepinephrine, t-ACPD, or bombesin. The responsiveness to amyloid β protein returned, subsequent to a period of resensitization for the individual agonists. It is suggested that this observed amyloid β protein stimulation of phospholipase C may be responsible for the elevated quantity of inositol seen in the brains of Alzheimer's disease patients.     

Stress-Activated Protein Kinase/c-Jun N-Terminal Kinase Phosphorylates τ Protein

Abstract: A proportion of the neuronal microtubule-associated protein (MAP) τ is highly phosphorylated in foetal and adult brain, whereas the majority of τ in the neurofibrillary tangles of Alzheimer's patients is hyperphosphorylated; many of the phosphorylation sites are serines or threonines followed by prolines. Several kinases phosphorylate τ at such sites in vitro. We have now shown that purified recombinant stress-activated protein kinase/c-Jun N-terminal kinase, a proline-directed kinase of the MAP kinase extended family, phosphorylates recombinant τ in vitro on threonine and serine residues. Western blots using antibodies to phosphorylation-dependent τ epitopes demonstrated that phosphorylation occurs in both of the main phosphorylated regions of τ protein. Unlike glycogen synthase kinase-3, the c-Jun N-terminal kinase readily phosphorylates Thr²⁰⁵ and Ser⁴²², which are more highly phosphorylated in Alzheimer τ than in foetal or adult τ. Glycogen synthase kinase-3 may preferentially phosphorylate the sites found physiologically, in foetal and to a smaller extent in adult τ, whereas stress-activated/c-Jun N-terminal kinase and/or other members of the extended MAP kinase family may be responsible for pathological proline-directed phosphorylations. Inflammatory processes in Alzheimer brain might therefore contribute directly to the pathological formation of the hyperphosphorylated τ found in neurofibrillary tangles.     

Activation of m1 Muscarinic Acetylcholine Receptor Regulates τ Phosphorylation in Transfected PC12 Cells

Abstract: Hyperphosphorylated τ proteins are the principal fibrous component of the neurofibrillary tangle pathology in Alzheimer's disease. The possibility that τ phosphorylation is controlled by cell surface neurotransmitter receptors was examined in PC12 cells transfected with the gene for the rat m₁ muscarinic acetylcholine receptor. Stimulation of m₁ receptor in these cells with two acetylcholine agonists, carbachol and AF102B, decreased τ phosphorylation, as indicated by specific τ monoclonal antibodies that recognize phosphorylation-dependent epitopes and by alkaline phosphatase treatment. The muscarinic effect was both time and dose dependent. In addition, a synergistic effect on τ phosphorylation was found between treatments with muscarinic agonists and nerve growth factor. These studies provide the first evidence for a link between the cholinergic signal transduction system and the neuronal cytoskeleton that can be mediated by regulated phosphorylation of τ microtubule-associated protein.     

Does dysregulation of the Notch and wingless/Wnt pathways underlie the pathogenesis of Alzheimer's disease?

Alzheimer's disease is characterized by the presence of neurofibrillary tangles and senile neuritic plaques in the brain. Tangles are aggregates of paired helical filaments composed of the microtubule-associated protein, tau, in a hyperphosphorylated state. Senile plaques have a core of amyloid beta-peptide derived by proteolysis of the amyloid precursor protein. A major hurdle in defining the pathogenic mechanisms in Alzheimer's disease is to understand how both amyloid beta-peptide deposition and paired helical filament formation are biochemically linked. Recent genetic discoveries provide some clues, suggesting that components of two developmentally important signalling pathways, Notch and wingless, or the vertebrate homologue of wingless, Wnt, are involved.     

Protein Kinase FA/Glycogen Synthase Kinase-3 Predominantly Phosphorylates the In Vivo Site Thr97-Pro in Brain Myelin Basic Protein: Evidence for Thr-Pro and Ser-Arg-X-X-Ser as Consensus Sequence Motifs

Abstract: In a previous study, protein kinase F_A/glycogen synthase kinase-3 ( F_A/GSK-3 ) was identified as a myelin basic protein (MBP) kinase associated with intact brain myelin. In this report, the phosphorylation sites of MBP by kinase F_A/GSk-3 were further determined by two-dimensional electrophoresis/TLC, phosphoamino acid analysis, tryptic peptide mapping, Edman degradation, and direct sequencing. Kinase F_A/GSK-3 phosphorylates MBP on both threonine and serine residues. Three tryptic phosphopeptide peaks were resolved by C₁₈ reverse-phase HPLC. Sequential manual Edman degradation together with direct sequence analysis revealed that T(p)PPPSQGK is the phosphorylation site sequence for the first major phosphopeptide peak. When mapping with the bovine brain MBP sequence, we finally demonstrate Thr⁹⁷-Pro, one of the in vivo phosphorylation sites in MBP, as the major site phosphorylated by kinase F_A/GSK-3, implicating a physiologically relevant role of F_A/GSK-3 in the regulation of brain myelin function. By using the same approach, we also identified NIVT⁹⁴(p)PR as the phosphorylation site sequence in the second major tryptic phosphopeptide derived from [³²P]MBP phosphorylated by kinase F_A/GSK-3, further indicating that kinase F_A/GSK-3 represents a Thr-Pro motif-directed MBP kinase involved in the phosphorylation of brain myelin.     

Chronic Ethanol Reduces Immunologically Detectable Gqα/11α in NG108–15 Cells

Abstract: The amyloid protein (βA₄) is found in the CNS of patients with Alzheimer's disease; however, the pathogenic role of this protein is not known. In the present study, a peptide fragment of βA₄βA₄ 25–35; Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-NH₂), which contains the conserved C-terminal sequence of substance P (X-Gly-Leu-Met-NH₂), and the neuropeptide substance P (SP) were examined for their ability to modulate nicotine-evoked secretion from cultured bovine adrenal chromaffin cells. Secretion of the released endogenous catecholamines was monitored by electrochemical detection after separation by HPLC. Secretion induced by 10⁻⁵ M nicotine was inhibited by SP and βA₄ 25–35. The IC₅₀ of SP and βA₄ 25–35 was 3 × 10⁻⁶ and 3 × 10⁻⁵ M , respectively. SP and βA₄ 25–35 both protected against nicotinic receptor desensitization. However, βA₄ 25–35 was ∼ 10-fold less effective than SP in its protective effect. The present work shows that βA₄ 25–35 can mimic the modulatory actions of SP on the nicotinic response of cultured bovine chromaffin cells, i.e., inhibition of the nicotinic response and protection against nicotinic desensitization. These modulatory actions may be associated with changes in nicotinic receptor levels reported to occur in Alzheimer's disease.     

τ Protein Kinase II Is Involved in the Regulation of the Normal Phosphorylation State of τ Protein

Abstract: To study the phosphorylation state of τ in vivo, we have prepared antisera by immunizing rabbits with synthetic phosphopeptides containing phosphoamino acids at specific sites that are potential targets for τ protein kinase II. Immunoblot experiments using these antisera demonstrated that τ in microtubule-associated proteins is phosphorylated at Ser¹⁴⁴ and at Ser³¹⁵. Almost all τ variants separated on two-dimensional gel electrophoresis were phosphorylated at Ser¹⁴⁴ and nearly one-half of them at Ser³¹⁵. Phosphorylation at Ser¹⁴⁴ and at Thr¹⁴⁷ of τ isolated from heat-stable brain extracts was shown to be developmentally regulated, with the highest level of phosphorylation found at postnatal week 1. In vitro phosphorylation of τ by τ protein kinase I, a kinase responsible for abnormal phosphorylation of τ found in paired helical filaments of patients with Alzheimer's disease, was enhanced by prior phosphorylation of τ by τ protein kinase II. Thus, we suggest that τ protein kinase II is indirectly involved, at least in part, in the regulation of the phosphorylation state of τ in neuronal cells.     

Selective Aggregation of Endogenous β-Amyloid Peptide and Soluble Amyloid Precursor Protein in Cerebrospinal Fluid by Zinc

Abstract: Zinc added to buffered solutions of synthetic β-amyloid peptide (Aβ) has been reported to induce accelerated formation of insoluble aggregates. This observation suggests that zinc may play a role in the formation of senile plaques, which contain Aβ, in Alzheimer's disease. To test this hypothesis under conditions more representative of the brain, we investigated the ability of zinc to induce aggregation of Aβ in freshly drawn canine CSF, which contains the same sequence as human Aβ. Aggregates were separated from CSF by ultracentrifugation before and after incubation with zinc and assayed by quantitative western blotting and ELISA. We found that zinc induced the rapid aggregation of endogenous Aβ in CSF, with an EC₅₀ of 120–140 µ M . The reaction was specific, because most (≥95%) CSF protein remained soluble under conditions where most Aβ was insoluble, as assayed by scanning densitometry of Coomassie-stained gels. Staining of the precipitated material resulted in the visualization of punctate regions that were thioflavin positive or birefringent when stained with Congo red, suggesting the formation of amyloid-related structures. These results suggest that zinc could play a role in amyloid deposition, because there is overlap between the regions of the brain where zinc concentrations are highest and regions with the highest amyloid content. It is surprising that zinc induced the aggregation of endogenous soluble APP at lower concentrations than required for Aβ (EC₅₀ 80 µ M ). The possibility that zinc-induced aggregation of APP may precede the deposition of Aβ into plaques is discussed. Investigation of aggregation of Aβ in CSF will aid in assessing the biological relevance of other agents that have been reported to accelerate amyloid formation.     

Acetylcholinesterase Is Increased in the Brains of Transgenic Mice Expressing the C-Terminal Fragment (CT100) of the β-Amyloid Protein Precursor of Alzheimer's Disease

Abstract: Acetylcholinesterase (AChE) expression is markedly affected in Alzheimer's disease (AD). AChE activity is lower in most regions of the AD brain, but it is increased within and around amyloid plaques. We have previously shown that AChE expression in P19 cells is increased by the amyloid β protein (Aβ). The aim of this study was to investigate AChE expression using a transgenic mouse model of Aβ overproduction. The β-actin promoter was used to drive expression of a transgene encoding the 100-amino acid C-terminal fragment of the human amyloid precursor protein (APP CT100). Analysis of extracts from transgenic mice revealed that the human sequences of full-length human APP CT100 and Aβ were overexpressed in the brain. Levels of salt-extractable AChE isoforms were increased in the brains of APP CT100 mice. There was also an increase in amphiphilic monomeric form (G^A₁) of AChE in the APP CT100 mice, whereas other isoforms were not changed. An increase in the proportion of G^A₁ AChE was also detected in samples of frontal cortex from AD patients. Analysis of AChE by lectin binding revealed differences in the glycosylation pattern in APP CT100 mice similar to those observed in frontal cortex samples from AD. The results are consistent with the possibility that changes in AChE isoform levels and glycosylation patterns in the AD brain may be a direct consequence of altered APP metabolism.